Principle of measurement and probe characteristics

Usually, it is not difficult to quantify an analyte in a test tube using a fluorescent probe for detection. In contrast, it is difficult to measure the intracellular concentration of the analyte, because there is usually no simple method to determine the concentration of the analyte in the environment of the cell independent of probe concentration.

Based on a intramolecular FRET mechanism (FRET = fluorescence resonance energy transfer), QuicGSH3.0 has been applied for measuring the intracellular GSH concentrations regardless of the probe concentrations. QuicGSH3.0 is a molecule formed with an orange fluorescent dye (T) and a deep-red fluorescent dye (S). At a low GSH concentration, green light excitation of T, leads to an energy transfer to S and a subsequent emission of deep-red fluorescent light. At a high GSH concentration, the energy transfer hardly occurs, so the probe emits the orange fluorescent light from the emission of T.

The mechanism enables determination of intracellular GSH concentration from a ratio of fluorescence intensities between the two wavelengths, regardless of the probe concentrations. QuicGSH3.0 has an optimum dissociation constant to GSH (Kd = 3.0 mM) for quantifying intracellular GSH concentrations which has been considered to be in a millimolar range. Because the ratio rapidly changes upon the GSH concentration change, QuicGSH3.0 can be used for real-time monitoring of intracellular GSH concentration changes.

Absorption and fluorescence spectra

Absorbance spectra (left) and emission spectra (right) of QuicGSH3.0

The emission maximum (λem) is 625 nm in the absence of GSH, whereas it shifts to 582 nm upon addition of GSH. Upon excitation with a green light at 520-550 nm, the ratio of fluorescence intensities between 582 nm (Fluorescent wavelength 1) and 625 nm (Fluorescent wavelength 2) changes as the GSH concentration changes.

An example of live-cell imaging

2 µM QuicGSH3.0 (containing 0.01% Pluronic F-127) was added to A549 cells and incubated for 10 minutes to be incorporated. The intracellular concentration of GSH (right) was calculated from images captured in the two wavelength ranges.

Refer the protocol document, detailed protocols for customers, and references for the details.